Corneal scarring severely limits vision for millions worldwide and is a contributing factor for the more than 40,000 corneal transplants in the USA annually. Although there are a wide variety of causes of corneal scarring, a common pathological feature is a marked alteration of corneal proteoglycans, a specialized group of extracellular matrix proteins modified with carbohydrate. The stromal proteoglycans play a key role in organizing the transparent extracellular matrix. There is compelling evidence that altered proteoglycans in scars contribute to loss of this essential organization. The long-term goals of this project are to elucidate the biological functions of proteoglycans in the cornea and determine how they contribute to corneal pathology. The continuation of this project will focus on the biosynthesis and biological functions of stromal glycosaminoglycans (GAGs), sulfated polysaccharides linked to proteoglycan proteins. The unique molecular composition of GAGs of normal transparent cornea is notably lost in scar tissue. The enzymes required for synthesis of these GAGs, however, and the genes regulating the expression of those enzymes are poorly understood. In addition, GAGs in many tissues regulate a variety of cellular functions by interaction with cell- surface receptors. In the cornea such interactions have not been characterized. The project will address these gaps in our understanding of these important molecules in the form of three Specific Aims, each formulated as a hypothesis. Aim 1 hypothesis: Abundance of specific glycosyltransferase and sulfotransferase gene transcripts regulates keratan sulfate synthesis in the cornea. Aim 2 hypothesis: GAG biosynthesis by keratocytes is mediated by cell-junction associated signaling. Aim 3 hypothesis: Corneal GAGs play a direct role in cellular events of inflammation and wound healing. The experimental approach uses cultures of primary keratocytes from bovine stroma, cells which accurately mimic matrix transitions observed in normal and pathological corneas. These in vitro experiments will be paired with in vivo murine models of non-inflammatory corneal wound healing and of corneal inflammation using wild-type mice and mice lacking genes for GAG biosynthetic enzymes. These experiments will produce results elucidating specific aspects of the synthesis and cellular control of corneal proteoglycans and, in addition, will generate novel insights on how interactions between corneal cells and the surrounding matrix regulate responses to wound healing and inflammation. PUBLIC HEALTH RELEVANCE. The proposed study will contribute to our long-term understanding of the molecular mechanisms regulating corneal scarring. It will add new insights into how corneal cells make scar tissue and how that tissue, in turn, influences the behavior of corneal cells. We expect this project ultimately to allow us to identify and manipulate molecular targets by which we can influence the composition of the stromal matrix, leading to therapeutic approaches to block or reverse corneal scarring without surgery.